Use of avarol for the control of adult T-cell leukemia/lymphoma

ABSTRACT

The invention concerns the use of avarol and its derivatives and pharmaceutical compositions thereof for the control of adult T-cell leukemia/lymphoma.

This is a continuation-in-part of prior-filed copending application Ser.No. 820,440, filed 1/17/86.

The present invention relates to the use of avarol and derivativesthereof for the control of adult T-cell leukemia/lymphoma.

Avarone and its hydroquinone derivative (avarol) are natural substanceswhich are present in the marine sponge Dysidea avara¹. Federal Republicof Germany OS 34 27 383, published Jan. 30, 1986, describes that avaroneand avarol and its derivatives have antitumoral, antibacterial andantimycotic properties which make them appear suitable, in particular,for the treatment of cancer and infectious diseases. Furthermore, it isstated therein that these compounds have a certain virostatic action invitro against cultures of herpes simplex virus and therefore against DNAviruses. However, no data has been heretofore available concerning thepossible inhibition of RNA viruses.

In addition to this, it has been found that avarone and its derivativeshave antimutagenic activity². Finally, it has been discovered thatavarone and its derivatives also have an antileukemic action³. Theexistence of this antileukemic action has been derived from studies invitro as well as in vivo with mouse leukemia cells L5178-Y. Up to thepresent time, however, no connection is known between such tumor cellsand adult T-cell leukemia and the virus which is causative of the same.Therefore, it was not obvious to use avarone, avarol, or the derivativesthereof for the control of the adult T-cell leukemia/lymphoma.

The object of the present invention is to provide a promising method ofcontrolling adult T-cell leukemia/lymphoma.

The adult T-cell leukemia (ATL)/lymphoma disease is caused through theattack of T-cells by the human T-cell-lymphotropic virus of type I(HTLV-I; RNA-virus). ATL is endemic in southwestern Japan, in theCaribbean, and in central Africa. For example, in Japan, ten percent tofifteen percent of the adults are infected with HTLV-I, as a result ofwhich each year one hundred to two hundred persons become ill with adultT-cell leukemia, of which more than one-half eventually die within ayear. An effective treatment of the adult T-cell leukemia has not beenavailable up to the present time.

It has surprisingly now been found that avarone or avarol and theirderivatives are excellently suited for controlling adult T-cellleukemia/lymphoma.

The object of the invention therefore is the use of avarol and itsderivatives of the general formula (Ib): ##STR1## and the 3,4-dihydroderivatives thereof, in which formulas R¹ and R² represent,independently of each other, a hydrogen atom, or a C₁ -C₄ alkylaminogroup,

R³ is a hydrogen atom, a C₁ -C₄ alkyl group, or a C₂ -C₆ acyl group, ora physiologically easily hydrolyzable ether or ester group, or both R³groups together stand for a C₄ -C₆ diacyl group,

and the physiologically compatible salts thereof, or a mixture of saidcompounds, for the control of adult T-cell leukemia/lymphoma.

Of the above-indicated compounds, avarol (formula Ib: R¹ =R² =R³ =H) ispreferably used. In the compounds of general formula (Ia), one of themoieties R¹ and R² is preferably a (Ib) both moieties R¹ and R² arepreferably a hydrogen atom.

The compounds of formula (Ib), which may contain amino groups, can beused as acid addition salts. There are concerned here salts withordinary physiologically compatible inorganic or organic acids such ashydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, lacticacid, maleic acid and the like.

The compounds of formula (Ib) in which R³ stands for H can also be usedas salts with ordinary physiologically compatible inorganic or organicbases such as sodium hydroxide, alkylamines, hydroxyalkylamines, and thelike.

Physiologically easily hydrolyzable esters and ethers of avarol and thecompounds of formula (Ib) in which R³ =H can easily hydrolyzeenzymatically or chemically in the body to the correspondinghydroquinone compounds. Suitable esters are, for example, theorthoformate acid ester, esters of alpha-ketocarboxylic acids, forinstance, pyruvic acid, toluene sulfonic acid esters and the like.Suitable ethers are, for instance, methoxymethylether,tetrahydropyranylether, and the like.

The preparation of avarol is described in Federal Republic of Germany OS34 27 383. For this the marine sponge Dysidea avara is extracted withethyl acetate. Avarol is recovered from the extract by columnchromatography on silica gel.

The derivatives of general formula (Ib) are prepared by reacting avarolor its alkylamino derivatives which are protected at the amino groupwith a C₂ -C₆ acyl chloride or with a corresponding carboxylicanhydride. As acyl chlorides use may be made, for instance, ofstraight-chain acyl chlorides, such as acetyl-, propionyl-, n-butyryl-,n-valeroyl-, and capronoyl chloride, as well as branched-chain acylchlorides, such as isobutyryl-, isovaleroyl-, 2-methylbutyryl- andtrimethylacetyl chloride. As suitable acid anhydrides there enter intoconsideration, for instance, straight-chain acid anhydrides such asacetanhydride, propionic anhydride, butyric anhydride, valericanhydride, and other carboxylic acid anhydrides, as well asbranched-chain acid anhydrides such as isobutyric anhydride, isovalericanhydride, 2-methylbutyric anhydride and trimethylacetanhydride. When aC₄ -C₆ -dicarboxylic acid chloride or an anhydride of a C₄ -C₆dicarboxylic acid is used, the cyclic esters of formula (Ib) areobtained.

The compounds of formula (Ib) are preferably reacted with the acylchlorides or carboxylic acid anhydrides in the presence of pyridine (S.de Rosa, L. Minale, R. Riccio and G. Sodano, J. Chem. Soc. Perkin I.1976, 1408-1414; Organikum, VEB Deutscher Verlag der Wissenschaften,13th edition, Berlin 1974, pages 441-446).

The C₁ -C₄ alkyl ethers of avarol and the compounds (Ib) can be preparedby reacting the hydroquinone compound, preferably in the form of the Naor K salt thereof, with the corresponding C₁ -C₄ alkyl halide or di-C₁-C₄ -alkyl-sulfate.

The compounds which are to be used in accordance with the invention aregenerally employed in the form of pharmaceutical compositions which areproduced in the form of dosage units and can be administeredsystemically, i.e., orally, rectally, or parenterally (intramuscular,intravenous and subcutaneous).

The compositions contain at least one compound of general formula (Ib)or a physiologically compatible salt thereof in an amount effective forthe treatment, elimination, alleviation, or improvement of adult T-cellleukemia/lymphoma, possibly together with a pharmaceutically compatibleexcipient and/or adjuvant. Such pharmaceutical agents contain, forinstance, 0.5 to 98 wt% of at least one compound of the invention,together with a pharmaceutical excipient.

If the agent is present in the form of a dosage unit, it preferablycontains 10 to 100 mg of the compound used in accordance with theinvention.

The pharmaceutical agents can be present, for oral administration insolid form, for instance, as tablets, pastilles, capsules or powder, orin liquid form, for instance, as aqueous or oil suspensions, syrup,elixir, solution or liquid-filled capsules.

Preferred oral agents are in the form of tablets or capsules and cancontain ordinary excipients such as binders (for instance, syrup,acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), fillers(for instance, lactose, sugar, corn starch, potato starch, calciumphosphate, sorbitol or glycine), lubricants (for instance, magnesiumstearate, talc, polyethylene glycol or silica), disintegration agents(for instance, starch) and wetting agents (for instance, sodium laurylsulfate).

Agents for parenteral administration are in general in the form of asolution or suspension of the compounds used in accordance with theinvention together with ordinary pharmaceutical excipients, forinstance, in the form of an aqueous solution for intravenous injectionor of an oil suspension for intramuscular injection. Agents suitable forparenteral administration are obtained by dissolving 0.1 to 10 wt% ofthe compounds of the invention in water or an excipient which consistsof an aliphatic polyalcohol such as glycerin, propylene glycol orpolyethylene glycols or mixtures thereof. The polyethylene glycolsconsist of a mixture of non-volatile, ordinarily liquid polyethyleneglycols which are soluble both in water and in organic liquids and themolecular weights of which range from 200 to 1500.

Pharmaceutical agents for rectal administration are in the form ofsuppositories, the compounds of the invention being incorporated in asuitable suppository base such as cocoa butter, hydrogenated fats,polywaxes or polyethylene glycols, in an amount of 1 to 10% by weight.

The pharmaceutical agents are prepared by ordinary methods, forinstance, by tabletting, incorporating of the compounds used inaccordance with the invention in a suppository base, sterile filtrationand filling in ampules or dropper bottles of a solution of the compoundsused in accordance with the invention in water for injection togetherwith ordinary additives such as sodium chloride, sodiumdihydrogenphosphate, disodium edetate (ethylene diaminotetraacetic aciddisodium salt), benzyl alcohol or sodium hydroxide in order to adjustthe pH.

The procedure for the treatment of adult T-cell leukemia/lymphomacomprises the administration of a therapeutically (antitumor) activeamount of avarol or a derivative thereof or mixtures thereof or of apharmaceutically-compatible salt thereof to a patient who requires thistreatment.

The dose depends primarily on the specific form of administration andthe purpose of the therapy. The size of the individual doses as well asthe program for administration can be determined best on basis of anindividual evaluation of the specific case by the doctor, in whichconnection the age, weight and condition of the patient, the route ofadministration and the nature and severity of the illness must be takeninto account. In general, the daily dose is 1 to 1000 mg and preferably10 to 500 and particularly 50 to 500 mg.

The duration of the treatment depends on the nature and severity of thedisease. It extends in general over several weeks, for instance, 4 to 8weeks.

The compounds to be used in accordance with the present invention act ina diversified manner against the human T-cell leukemia virus of type I(HTLV-I) and the T-cells attacked by the virus (having both antiviraland antitumor activity). These compounds can therefore be employed forthe treatment of diseases caused by the HTLV-virus.

The antiviral and antitumor activity of the compounds used in accordancewith the invention will be examined below on basis of pharmacologicalin-vitro test systems using avarone as example.

For this, the MT-2, ATL-3I, and ATL-1K cell lines were employed. Thesecell lines are described by Hashino, H., et al., Proc. Nat. AcademyScience U.S.A. 80, 6061-6065 (1983) and by Miyoshi, J., et al., Nature294, 770-771 (1981). All these cell lines contain the HTLV-provirus, andamong these cell lines primarily the lines MT-2 and ATL-3I express alsothe retrovirus antigens and also produce the HTLV-I virus.

TEST PROCEDURE

Purification of HTLV-I reverse transcriptase and assay conditions:

The HTLV-I reverse transcriptase used for the present experiments(prepared by the method of P. S. Sarin, Y. Taguchi, D. Jun, A. Thornton,R. C. Gallo, B. Oeberg, Biochem. Pharmacol. 34, 1985, pages 4075 to4079) was purified by sequential chromatography on DEAE-cellulose,phosphocellulose, and hydroxyapatite. The purified enzyme was stored in50 mM tris-HCl (trishydroxymethylaminomethane) (pH 7.5), 1 mMdithiothreitol (DTT), 0.01% Triton X-100 and 20% glycerin. The assaysfor reverse transcriptase were carried out in a reaction mixture (50 μl)which contained 50 mM tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl₂, 100 mMpotassium chloride, 0.01% Triton X-100 (C₈ H₁₇ -C₆ H₄ -(OCH₂ CH₂)₉₋₁₀-OH) or NP40 (non-ionic detergent Nonidet P 40, brand name of the Sigmacompany, i.e. octylphenol ethylene oxide condensate), 10 μg/ml(dT)₁₅.(A)_(n) (hybrid polymers of the oligo- or polynucleotidesoligodeoxythymidylic acid and polyadenylic acid) as template primer and[³ H]-deoxythymidine triphosphate ([³ H]-dTTP). The reaction mixture wasincubated for one hour at 37° C. and the reaction was stopped by adding50 μg of yeast-tRNA and 2 ml of a 10% trichloroacetic-acid solution(TCA) containing 1 mM of sodium pyrophosphate. The samples were filteredthrough a millipore filter (0.45 μm), and washed, first with 5%TCA-solution (5x) and then with 2 ml of 70% ethanol. The filters weredried under a heating lamp, whereupon scintillation liquid was added andthe radioactivity was determined in a beta-scintillation counter.

CELL CULTURE CONDITIONS

Cell culture conditions were employed according to those described by H.Hoshino, et al., Proc. Nat. Acad. Sci. (U.S.A.) 80, 1983, 7337-7341.

IMMUNOFLUORESCENCE ASSAY

The immunofluorescence assays were carried out on methanol:acetone(1:1)-fixed ATL-3-I cells with the use of a standardized ATL-patientserum against the retrovirus antigen (code 3 KI, H. Hoshino et al., seeprevious literature citation). The ATL-3-I cells with or without drugtreatment were fixed on toxoplasmosis slides. After fixation withmethanol-acetone (1:1) for 30 minutes at room temperature, the slideswere stored in sealed plastic containers at -20° C. until use. TheATL-patient serum was added to the cells, incubated at room temperaturein a moisture chamber for one hour and washed for two hours with PBS(phosphate buffered saline) containing 0.25% Triton X-100. The cellswere then treated for one hour with fluorescein (FITC) labeled rabbitanti-human IgG (Capell Labs.) and washed overnight with PBS buffercontaining 0.25% Triton X-100. Fifty percent glycerin was added to theslides and the cell fluorescence was determined with a Zeissfluorescence microscope.

1. Cell Growth

ATL-3-I, MT-2, and ATL-1K cells were seeded into Petri dishes in aconcentration of 3×10³ cells/cm² in a culture medium containing 20% ofcalf serum. After incubation for two days, the density of the cellsamounted to: AMT-3-I: 13.2×10³ cells/cm² ; MT-2: 12.4×10³ cells/cm² ;ATL-1K: 15.7×10³ cells/cm². These values formed the control values.

Thereupon samples of these cells were treated for two days withdifferent concentrations of avarol. The following results were obtained:

    ______________________________________                                                           Cell concentration ×                                         Concentration of                                                                         10.sup.3 /cm.sup.2                                                 the drug (μg/ml)                                                                      ATL-3-I  MT-2    ATL-1K                                    ______________________________________                                        Control   0            13.2     12.4  15.7                                    infected                                                                      Avarol    1.0          9.4      8.1   13.4                                    (infected cells)                                                                        5.0          8.7      7.9   10.3                                    ______________________________________                                    

It is clear that avarol inhibited the growth rate of ATL cells.

2. Inhibition of the Production of Reverse Transcriptase by ATL-3-Icells which were treated with Avarone or Avarol

It was examined whether the two-day addition of avarol to ATL-3-I cellsstops the production of HTLV-I viruses. Reverse transcriptase wasselected as measure for the amount of virus in the culture medium.Therefore, inhibition of the reverse transcriptase indicates inhibitionof the production of virus. The results are set forth in the followingtable:

    ______________________________________                                                      Concentration                                                                              Reverse                                                          of the compound                                                                            Transcriptase                                      Compound added                                                                              (μg/ml)   activity (in %)                                    ______________________________________                                        None          --           100                                                Avarol        1            83                                                               5            52                                                 ______________________________________                                    

It is clear that, in the supernatant of the ATL-3-I cells which were nottreated with avarol, a considerable activity of reverse transcriptasewas indicated. The addition of avarol led to a dose-dependent reductionof the reverse transcriptase activity in the supernatant. The compoundsused in accordance with the invention are therefore able to inhibitvirus replication practically completely at a dose of 5 μg/ml.

3. Inhibition of Retroviral (HTLV-I) Antigen Expression in ATL-3-I byAvarol

It was found that avarol possesses a strongly inhibitory action on theexpression of retroviral antigen in ATL-3-I cells. When the ATL-3-Icells were cultivated without the compound to be tested, there wasexpression of the retroviral antigen. After incubation of the ATL-3-Icells with the compound to be tested, a strong protective effect wasobserved. The following results were obtained:

    ______________________________________                                                     Concentration                                                                              Expression of                                                    of the compound                                                                            retroviral antigen                                  Compound added                                                                             (μg/ml)   (in %)                                              ______________________________________                                        None         --           100                                                 Avarol       1            82                                                               5            74                                                  ______________________________________                                    

It is clear that avarol caused a significant reduction in the expressionof the retroviral antigen.

Toxicity

The in-vivo toxicity (mg compound/kg) of avarol in male NMRI mice is asfollows:

Acute toxicity: LD₅₀ (269.1), LD₁₀ (156.4) Subacute toxicity: LD₅₀(218.4), LD₁₀ (138.6) (Muller et al., Cancer Research 1985, 45,4822-4826).

IDENTITY

The active antiviral and antitumor ingredients or agents of the presentinvention have the formulas:

Avarol2-[(1R)-1,2,3,4,4a,7,8,8aα-octahydro-1β,2β,4aβ,5-tetramethyl-1-naphthylmethyl]-1,4-benzenediol##STR2## Molecular Formula of Avarol

C₂₁ H₃₀ O₂ ; Mol wt: 314.22 C 80.32%; H 9.81%; O 9.87% and the3,4-dihydro derivatives thereof.

The compound 3,4-dihydroavarol is known from L. Minale et al.,Tetrahedron Letters 36, 3401-3404 (1974) and Muller et al., CancerResearch 45, 4822-4826 (1985).

PRODRUGS OR PRECURSORS AND THEIR PREPARATION

The compound avarol and its 3,4-dihydro derivatives may also be employedor embodied in pharmaceutical compositions according to the inventionand administered in the form of compounds which convert or metabolizethereto after introduction into the living animal body. Such compoundsare commonly referred to today as prodrugs or precursors, andrepresentative examples include their esters and alkylamino derivatives.As already indicated, some of these compounds are known in the priorart, whereas others are made in a known manner corresponding thereto.Representative of such prodrugs and precursors, and their preparation,are set forth in the foregoing and in the following.

The following Examples serve to explain the invention, but are not to beconstrued as limiting.

EXAMPLE 1 Avarone and avarol

3 kg of fresh sponge (water containing) are crushed in a Starmix™ mixerand extracted with 250 ml of ethyl acetate; the extract thus obtained isdried over magnesium sulfate and then filtered. The filtrate isconcentrated to dryness. The remaining residue (about 50 g) is taken upin about 100 ml of benzene and chromatographed over a silica-gel column(about 200 g) with benzene as eluent. Avarone appears in the eluatewhile avarol remains in the column. Avarol is eluted with a mixture ofbenzene and ethyl acetate (90:10, V:V). The eluate is concentrated todryness and avarol then obtained in pure form by crystallization fromdichloromethane-acetone. Avarone is purified by recrystallization frombenzene. Yield: 0.7 g avarone; 8.9 g avarol; avarone MP: 62°-64° C.;avarol MP: 148°-150° C.

EXAMPLE 2 Avarol diacetate

(a) 500 mg of avarol are dissolved in 20 ml of absolute pyridine and 1 gof acetyl chloride is added in individual portions to the solution,while shaking. The solution is worked up in the customary manner,concentrated to dryness, and the residue extracted with boiling heptane.Upon cooling, the ester crystallizes out. It is recrystallized fromhexane; MP: 62°-64° C.; yield about 430 mg.

In similar manner there were obtained:

(b) avarol dipropionate

(c) avarol divalerianate

(d) avarol ditrimethylacetate.

EXAMPLE 3 Avarol dicapronate

(a) 300 mg of avarol are dissolved in 25 ml of absolute pyridine and 0.6g of caproic anhydride added in separate portions to the solution whileshaking. It is worked up in the customary manner, concentrated todryness, and the residue extracted with boiling heptane. It isrecrystallized from acetone and then from hexane. Yield: 210 mg.

In similar manner there were obtained:

(b) avarol diisovalerianate

(c) avarol diethylmethylacetate

(d) avarol succinate.

EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

In the following examples of formulations there can be used as activesubstance in each case one of the compounds used in accordance with theinvention by itself or in mixture with another compound according to theinvention.

EXAMPLE A

    ______________________________________                                                      Tablet Formulation                                              ______________________________________                                        Active substance*                                                                             10          mg                                                Lactose         18          mg                                                Potato starch   38          mg                                                Gelatin         2           mg                                                Talc            2           mg                                                Magnesium stearate                                                                            0.1         mg                                                ______________________________________                                         *e.g., avarol, 3,4dihydroavarol, or prodrug or procursor thereof         

EXAMPLE B

    ______________________________________                                                      Tablet Formulation                                              ______________________________________                                        Active substance*                                                                             10 mg                                                         Potato starch   40 mg                                                         Polyvinylpyrrolidone                                                                           5 mg                                                         ______________________________________                                         The tablets are coated with a colored layer of sugar.                    

EXAMPLE C

    ______________________________________                                                     Capsule Formu1ation                                              ______________________________________                                        Active substance*                                                                            10 mg                                                          Corn starch    90 mg                                                          Lactose        50 mg                                                          Talc            2 mg                                                          ______________________________________                                         This mixture is introduced into gelatin capsules.                        

EXAMPLE D

    ______________________________________                                                      Injection Solution                                              ______________________________________                                        Active substance*                                                                             12 mg                                                         Sorbitol        40 mg                                                         Sterile water to                                                                               1 ml                                                         ______________________________________                                    

EXAMPLE E

    ______________________________________                                                            Liquid                                                                        Oral Formulation                                          ______________________________________                                        Active substance      2         g                                             Saccharose            250       g                                             Glucose               300       g                                             d-Sorbitol            150       g                                             Agar-agar             0.15      g                                             Methylparaben         0.5       g                                             Propylparaben         0.05      g                                             Flavoring substance (orange flavor)                                                                 10        g                                             Tartazin yellow                                                               Purified water to     1000      ml                                            ______________________________________                                    

EXAMPLE F

    ______________________________________                                                          Liquid                                                                        Oral Formulation                                            ______________________________________                                        Active substance    2          g                                              Tragacanth          7          g                                              Glycerin            50         g                                              Saccharose          400        g                                              Methylparaben       0.5        g                                              Propylparaben       0.05       g                                              Flavoring substance (flavor of                                                                    10         g                                              black currants)                                                               Red dye No. 2C.E. 184                                                                             0.02       g                                              Purified water to   1000       ml                                             ______________________________________                                    

EXAMPLE G

    ______________________________________                                                          Liquid                                                                        Oral Formulation                                            ______________________________________                                        Active substance    2.4        g                                              Saccharose          400        g                                              Tincture of bitter orange peels                                                                   20         g                                              Tincture of sweet orange peels                                                                    15         g                                              Purified water to   1000       ml                                             ______________________________________                                    

The following sections respectively evidence that Avarol is effective incombination with other active compounds or principles; that itsemployment has no adverse effect upon the immune response of a subjecttreated therewith; and that an advantageous treatment regimen can bedesigned because of their extended half-life.

APPLICATION OF AVAROL IN COMBINATION WITH OTHER COMPOUNDS

It is also advantageous to use Avarol in combination with othercompounds.

EXAMPLE

Combination studies were performed in vitro with Avarol together withdiethyldithiocarbamate (DDC). DDC has previously been shown to haveimmunomodulating activity (Lang et al., Lancet 2: 1066; 1985) in AIDSpatients and acts by inhibition of superoxide dismutase (SODase) both invitro and in vivo (Heikkila et al., J. Biol. Chem. 251: 2182-2185; 1976and Heikkila et al., In: Superoxide and Superoxide Dismutase (eds. A. M.Michelson, J. M. McCord and I. Friedovich), Academic Press, New York;pp. 367-373).

METHODS

L5178y mouse lymphoma cells were grown in roller tube cultures inEagle's minimum essential medium, supplemented with 10% horse serum(Muller et al., Cancer Res. 39: 1102-1107; 1979 and Muller et al. CancerRes. 45: 4822-4826; 1985). For the dose response experiments, 5 mlcultures were initiated by inoculation of 5000 cells/ml and incubated at37 degrees C. for 72 hours; the controls had generation times of10.4-10.6 hours. The cell growth was determined by cell count with acomputer supported cell-counter (128-channel counter; Cytocomp, systemMichaelis: BIOTRON-Medizinelektronik, Mainz; West Germany). The ED50(±SD) was estimated by logit regression (L. Sachs, Angewandte Statistik,Springer-Verlag Berlin 1984). The mathematical evaluation of thefractional inhibitory concentration indices (FIC indices) ofAvarol/Avarone-DDC combinations was performed according to publishedequations and experimental conditions (Muller et al., Cancer Lett. 1:127-132; 1976; and Phillips et al., Antimicrob. Agents Chemother. 9:736-740, 1976), FIC>1 are interpreted as antagonistic; FIC=1 as additiveeffects; FIC<1 as suggestive of synergism; and FIC<0.5 as significantsynergism.

RESULTS

In this set of experiments, L5178y cells were incubated with Avarol inthe absence or presence of the established SODase inhibitor DDC. In theabsence of DDC, the ED50 for avarol was determined to be 0.94±0.14 μM,as described previously (Muller et al., Cancer Res. 45: 4822-4826;1985).

Coaddition of DDC at the concentrations of 1.46 μM or 1.71 μM to thecultures, significantly lowered the ED50 concentrations to 0.27±0.04 μMand 0.108±0.015 μM. The two DDC concentrations chosen caused aninhibition of cell growth by 26 and 38% respectively in the absence ofAvarol.

As a further proof for the additive effect of Avarol in combination withDDC, the FIC indices were determined.

Concentration ratios between 0.2:1 to 1.3:1.0 (Avarol:DDC) were chosen.The calculated FIC indices varied between 0.82 and 1.04 indicating anadditive interaction between Avarol and DDC (Table).

CONCLUSION

These studies show that Avarol can be administered in vitro incombination with other therapeutic agents resulting in a beneficialtherapeutical effect.

                  TABLE                                                           ______________________________________                                        Fraction inhibitory concentration indices (FIC indices)                       for avarol-DDC combination on L5178y cells.                                   The standard incubation conditions (5 ml and 72 hours)                        were chosen. The combination ratios are based on x μM                      avarol/avarone to y μM DDC.                                                Drug combination                                                                            Concentration ratio                                                                         FIC index                                         ______________________________________                                        Avarol:DDC    0.3:1.0       0.87                                                            0.5:1.0       0.82                                                            1.3:1.0       0.90                                              ______________________________________                                    

INFLUENCE OF AVAROL ON SELECTED IMMUNE RESPONSES IN VITRO AND IN VIVO

In vitro studies: Previous studies revealed that T-cell derived leukemiacells are more sensitively inhibited by avarol than normal T- orB-lymophocytes (Muller et al., Cancer Res. 45: 4822-4826; 1985 andMuller et al., Eur. J. Cancer Clin. Oncol. 22: 473-476; 1986). Thelymphoma cell growth is inhibited by 50% within the concentration range0.3-0.4 μg/ml (=0.9-1.1 μM), while the [³ H] dThd-incorporation rate innormal T-lymphocytes is inhibited by 50% between 0.5-1.3 μg/ml and, innormal B-lymphocytes, between 1.6-1.9 μg/ml. Therefore the following invitro studies were performed with 0.5-3 μg/ml of avarol.

In vivo studies: From previous experiments with NMRI mice, bearingL5178y cells, it is known that the chemotherapeutical dose of avarol isin the range of 1-50 mg/kg at a 50% lethal dose of 269.1 mg/kg (Mulleret al., Cancer Res. 45: 4822-4826; 1985). The 50% lethal dose (treatmentfor 5 days) for male Sprague-Dawley rats was determined to be 235.7mg/kg. Therefore, daily doses of avarone of 1-30 mg/kg/injection werechosen for the following in vivo studies.

Rationale: Considering an application of avarol and avarone in thetreatment of acquired immunodeficiency syndrome [AIDS] patients andadult T-cell lymphoma/leukemia patients, the effect of avarol on immuneresponses was studied in vitro and in vivo. The results demonstrate thatavarol causes, at antileukemic doses in vivo, no influence ondelayed-type hypersensitivity (DTH) and on antibody-mediatedhypersensitivity (AMH) but displays a stimulatory effect on antibodyproduction both in vitro and in vivo. These properties are veryadvantageous, especially for AIDS patients, which show both B- andT-cell deficiencies (Ammann et al., J. Amer. Med. Ass. 251: 1447-1449;1984).

1. IN VITRO IMMUNOGLOBULIN (IG) PRODUCTION Method

Human peripheral blood lymphocutes (6×10⁵ cells/ml) were cultured in thepresence of 0 to 2 μg/ml of pokeweed mitogen (PWM; Sigma no 9379) inDulbeccos minimum essential medium, supplemented with 20% fetal calfserum, for 6 days. The assays were performed in a final volume of 200 μlin microtiter plates (Dynatech; M129B) (Muller et al., Eur. J. CancerClin. Oncol. 22: 473-476; 1986). Avarol in a concentration range of 0-3μg/ml was added at time 0 or 3 days after starting the culture. Thesupernatants were harvested and assayed for human immunoglobulincontent.

The culture supernatants were assayed by the enzyme-linked immunosorbentassay (Houtman et al., Clin. Exp. Immunol. 62: 696-704; 1985) usinganti-human IgG (Dakopatts, Hamburg; P214) and anti-human IgM (Dakopatts;P215). These antibodies were conjugated to peroxidase. Bound enzymeactivity was measured using 2,2'-azinobis(3-ethylbenthiazolinesulfonicacid) (Sigma no. A-1888) as substrate.

Results

Under the assay conditions used, human peripheral blood lymphocytes(unseparated) produced 1.3 μg/ml of IgG and 8.9 μg/ml of IgM during a6-day culture (Table 1). The production was enhanced upon addition ofPWM to 4.1 μg/ml (IgG) and 12.3 μg/ml (IgM). I used a PWM concentrationof only 2 μg/ml, which was previously found to be suboptimal, in orderto detect more sensitively a possible Ig production after avaroltreatment. The results revealed a significant increase of both IgG andIgM production in cultures treated with 0.3 or 1 μg/ml of avarol,irrespectively of the presence of PWM (Table 1) and of the time periodfor avarol presence (0 to 6 days or 3 to 6 days). At higher avarolconcentrations, the Ig production turned to normal values.

2. IN VIVO IMMUNOGLOBULIN (IG) PRODUCTION

Concerning the functional consequences of the compound onto B-cells invitro, I found (previous section) that both the IgG and the IgMsecretion is stimulated in cultures of human lymphoid cells(unseparated) which are treated with non-cytostatic concentrations ofavarol. This finding is supported by the results of the Jerne plaquetest. The experiments revealed that single-cell suspensions preparedfrom the spleens of mice, immunized with sheep red blood cells (SRBC),that had been pretreated with therapeutical doses of avarol, contained ahigher number of plaque-forming cells that the spleens from untreatedanimals.

Method

The plaque test of Jerne and Nordin (Science 140: 405-406, 1963) wasused with the modification given by Cunningham and Szenberg (Immunolog.14: 599-601; 1968). Briefly, 8 male NMRI mice per group (20-22 g) wereimmunized on day 0 by i.p. injection of 5×10⁸ SRBC in 0.5 ml saline.Treatment with avarol was done i.p. either from day 0 to day +4 or fromday -4 to day 0. One hour after the last drug administration, theanimals were killed by a blow on the head and bled. The spleens wereremoved and a single cell suspension was prepared by teasing the pooledspleens through a 120μm mesh sieve. After lysing the erythrocytes by0.15M NH₄ Cl, the cells were washed twice in Hank's balanced saltsolution containing 29 ml of 7.5% NaHCO₃ and 10 ml of 1M HEPES buffersolution per one liter. The cells were resuspended in 2 ml per spleen.Six Cunningham chambers per group were filled with a mixture of 0.5 mlof a suitable dilution of spleen cells (generally 1:100), 0.5 ml of asuspension of SRBC (10⁹ /ml) and 125 μl of normal guinea pig complement.After 90-120 min of incubation at 37° C., the number of plaques wascounted. Plaques per spleen or per 10⁶ spleen cells were calculated.

The pooled blood of 8 mice was centrifuged to obtain the serum. IgM andIgG titers were measured with the mercaptoethanol method (Hudson, L. andHay, F. C. Practical Immunology, Blackwell Sci., Oxford; 1983).

Results

Applying the modified Jerne assay the number of plaque forming cells inspleen of mice, injected with SRBC, was determined in the absence orpresence of avarol treatment (Table 2). Animals, treated with 30 mg/kgof avarol from day 0 to day +4, had a 28% higher number of plaqueforming cells/spleen, and those treated from day -4 to day 0, a 21%increase.

3. DELAYED-TYPE HYPERSENSITIVITY (DTH) Method

Reaction to sheep red blood cells (SRBC): DTH was produced according tothe method of Lagrange et al. (J. Exp. Med. 139: 528-542; 1974) and Liew(Eur. J. Immunol. 7, 714-718; 1977). Male NMRI mice (20-23 g, 10animals/group) were immunized with 10⁸ SRBC/40 μl into one foot pad (day0). Four days later 10⁹ SRBC/40 μl were injected into the other footpad. After 24 hours, paw size was measured with a dial gauge caliperOdi-Test OOT (H. C. Kroeplin, Schluchtern, Germany). Avarol (in 0.15%[w/v] methylcellulose) was administered daily i.p. either from day -2 to+1 or from day +2 to +4. The level of DTH was expressed as foot padincrease, determined after 24 hrs; and it is given in mm. Non-treatedgroups were injected i.p. with methylcellulose only. Treated groups werecompared with the controls using the U-test of Wilcoxon et al. (Sachs,L. Angewandte Statistik, Springer-Verlag, Berlin; 1984).

Contact allergy to oxazolone: Allergic contact dermatitis againstoxazolone was produced according to the method of Asherson and Ptak(Immunol. 15: 405-416; 1968) as well as those of Bure and Degrand(Agents and Actions 9: 534; 1979). Male NMRI mice (20-22 g; 10/group)were sensitized with 100 μl of a 2% oxazolone in acetone solution ontothe shaved abdomen (day 0). Animals were treated with avarol (i.p.)daily from day -1 to +2 or from day +6 to +8; control group was treatedwith deoxymethasone (Casella-Hoechst) as a standard per os at a dailydose of 30 mg/kg for the same period of time as for avarol. Seven daysafter sensitization 10 μl of a 3% oxazolone (this concentration waspreviously found to give more reproducible results than the lower 2%concentration) solution were topically applied onto the inner side ofone ear, whereas the other ear received only acetone. Twenty-four hourslater the animals were killed and two 8 mm (diameter) pieces werepunched out of both ears. The weight differences were recorded. Treatedgroups were compared with controls using the U-test.

Results

The effect of avarol on cell-mediated, or delayed type hypersensitivity,reaction in mice was determined in two ways; (i) reaction to sheep redblood cells and (ii) reaction to oxazolone sensitization.

Delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC):Avarol was administered at three different doses and during twodifferent time periods as described under "Methods". Given during theperiod -2 to +1 (with respect to the last SRBC administration), avarolcaused no significant immunosuppressive influence on DTH to SRBC in NMRImice up to daily doses of 30 mg/kg. However, when given on day +2 to +4after immunization, avarol administration at doses higher than 10 mg/kgresulted in a weak but significant immunosuppressive effect.

Contact allergy to oxazolone: In a second approach, the mice weretopically sensitized with oxazolone and challenged again after 7 dayswith the same irritant. Avarol or deoxymethasone (=negative control)were administered from day -1 to +2 or from day +6 to +8 with respect tothe day of sensitization, as described under "Methods". Deoxymethasonecaused a significant immunosuppressive influence on DTH reaction (24hours increase in ear thickness [in mg] if treated from day -1 to +2:10.2±6.0, or 10.9±6.1 at a treatment from day +6 to to +8; thecorresponding controls are 22.3±5.6 and 18.5±3.7, respectively). On theother hand, avarol in a dose range from 3-30 mg/kg either from day -1 to+2 or day +6 to +8 did not influence significantly P>0.05) the DTHoxazolone.

4. ANTIBODY MEDIATED HYPERSENSITIVITY (AMH) Method

A modified active Arthus reaction was performed as described (Titus etal., J. Immunol. Methods 45: 65-78; 1981). Male Sprague-Dawley rats(8/group) weighing 120 g were immunized by injecting into the tail base0.5 ml of a suspension of 4.4 ml pertussis vaccine (Behringwerke,Marburg) in 65.6 ml of 0.9% saline plus 0.7 g ovalbumin (3×cryst.) in100 ml paraffin oil. Three weeks after immunization 0.1 ml of 0.03%ovalbumin solution were injected into a hind paw and the paw volume wasmeasured with a water plethysmometer (Rhema) immediately thereafter andagain after 4 hours. The animals were treated i.p. with daily doses from3-30 mg/kg of avarol (in 0.15% [w/v] methylcellulose) according to thefollowing schedules: (i) day -1 to +2, (ii) day +18 to +21 or (iii)twice 24 hours and 30 minutes before challenge (day 0 represents the dayof immunization). The controls received methylcellulose only. Values arecompared with a control group using Student's-t-test (L. Sachs.Angewandte Statistik, Springer-Verlag, Berlin; 1984).

Results

I used a modified Arthus reaction to determine the possible influence ofavarol on the AMH in rats, caused by ovalbumin. The results revealed nosignificant suppression of the Arthus reaction by avarol, irrespectivelyof the following schedules chosen; (i) day -1 to +2 or (ii) day +18 to+21, related to day 0 of immunization or (iii) if applied immediatelybefore the challenge with ovalbumin. The weak suppression of thereaction, observed when rats were treated with avarol (30 mg/kg) 24hours and 30 minutes before challenge, was also found to be notsignificant (P>0.05).

In Summary

The effect of avarol on the lymphoid system was studied both in vitroand in vivo. Avarol increased significantly the IgG and IgM productionby cultures of human lymphoid cells (unseparated) in vitro and slightlythe number of plaque forming cells in vivo in spleen of mice. Moreover,a pretreatment of mice with the avarol resulted in a higher [³ H]-dThdincorporation rate in both macrophage-containing and macrophage-depletedlymphocyte cultures in vitro. The stimulatory influence of avarol onhumoral immune responses is not accompanied by a change of theantibody-mediated hypersensitivity reaction, as measured by the Arthusreaction. No significant influence of avarol on the cellular immunesystem in vivo (rats or mice) was found, as taken from studies ondelayed-type hypersensitivity reactions to sheep red blood cells and tooxazolone. The in vitro and animal data indicate that avarol combinesuseful properties, e.g. anti-HIV efficience in vitro and augmentation ofhumural immune responses.

                                      TABLE 1                                     __________________________________________________________________________    Mean immunoglobulin production in cultures of                                 peripheral human blood lymphocytes in dependence on the                       mitogenic effect of PWM and on avarol                                         Avarol     Immunoglobulin synthesized (μg/ml)                              concentration                                                                            IgG time (days)                                                                            IgM time (days)                                       (μg/ml)                                                                           PWM 0 to 6                                                                              3 to 6 0 to 6 3 to 6                                         __________________________________________________________________________    0      -   1.3 ± 0.3                                                                        1.4 ± 0.3                                                                          8.9 ± 2.0                                                                         8.5 ± 1.8                                         +   4.1 ± 0.7                                                                        4.0 ± 0.8                                                                         12.3 ± 2.6                                                                        11.7 ± 2.2                                  0.3    -   2.1 ± 0.5+                                                                       2.0 ± 0.5+                                                                        14.7 ± 3.4+                                                                       14.2 + 3.3+                                           +   6.7 ± 1.5+                                                                       7.9 ± 1.9++                                                                       20.4 ± 5.1+                                                                       24.7 ± 5.6++                                1.0    -   2.3 ± 0.6+                                                                       2.2 ± 0.5+                                                                        15.6 ± 4.0+                                                                       15.9 ± 4.2++                                       +   7.0 ± 1.6+                                                                       8.5 ± 1.9++                                                                       23.5 ± 5.5++                                                                      25.6 ± 5.9++                                3.0    -   1.2 ± 0.3                                                                        1.3 ± 0.3                                                                          8.1 ± 1.8                                                                         8.4 ± 1.9                                         +   3.9 ± 1.0                                                                        4.9 ± 1.0                                                                         10.5 ± 2.6                                                                        11.7 ± 2.9                                  __________________________________________________________________________     +P < 0.01;                                                                    ++P < 0.005                                                              

                                      TABLE 2                                     __________________________________________________________________________     Effect of avarol on the number of plaque                                     forming cells in the spleen of mice                                                       Cell/                                                             Avarol                                                                             Schedule                                                                             spleen                                                                             Plaque forming cells per:                                                                  Titer                                           (mg/kg)                                                                            (days) (× 10.sup.-7)                                                                spleen (× 10.sup.-5)                                                            10.sup.6 cells                                                                     IgM IgG                                         __________________________________________________________________________    0    from 0 to +4                                                                         11.3 3.13    2,782                                                                              256 4                                                from -4 to 0                                                                         11.6 2.62    2,259                                                                              512 4                                           30   from 0 to +4                                                                         12.0 3.98    3,316                                                                              256 2                                                from -4 to 0                                                                         14.4 3.18    2,202                                                                              512 4                                           __________________________________________________________________________

DETERMINATION OF THE HALF-LIFE OF TRITIUM-LABELLED AVAROL AND OF THEORGAN DISTRIBUTION RATS

    ______________________________________                                        Animals: Sprague-Dawley rats (male).                                          Half-life                                                                     Avarol was injected (intravenously) at therapeutic doses                      into rats. The following concentrations of Avarol were                        chosen: 2 mg/kg, 10 mg/kg, and 20 mg/kg.                                      Tritium labelled Avarol was obtained by converting Avarol                     into Dihydro-avarol in the presence of tritium gas.                           Percentage of Radioactivity in Blood                                          (100% is set as the amount of radio-                                          activity present 10 minutes after                                             termination of i. v. injection)                                                        Time of Determination (hours after                                   Doses of termination of i. v. injection)                                      Avarol   1         6      12       24   48                                    ______________________________________                                         2 mg/kg 95        91     73       41   23                                    10 mg/kg 96        88     72       43   26                                    20 mg/kg 92        83     75       39   21                                    ______________________________________                                    

From these determinations it is evident that the half-life of Avarol inrats is approximately 22 hours.

Organ Distribution

The organ distribution was determined by the same method using tritiumlabelled Avarol. The determinations were performed 24 hours aftertermination of the i.v. application.

    ______________________________________                                                              Concentration of Avarol                                          Doses of Avarol                                                                            (microgram of Avarol                                    Organ    (mg/kg)      per gram of tissue)                                     ______________________________________                                        Blood    2            4.3                                                              10           5.2                                                              20           5.8                                                     Spleen   2            1.7                                                              10           1.9                                                              20           2.2                                                     Liver    2            6.4                                                              10           6.9                                                              20           7.4                                                     Kidney   2            1.6                                                              10           2.2                                                              20           2.4                                                     Heart    2            0.7                                                              10           0.9                                                              20           0.9                                                     Testes   2            0.48                                                             10           0.63                                                             20           0.67                                                    Brain    2            0.45                                                             10           0.58                                                             20           0.64                                                    Skin     2            0.4                                                              10           0.6                                                              20           0.6                                                     ______________________________________                                    

These concentrations were determined on the basis of radioactivity.Moreover, the chemical nature of the radioactive product was identifiedby high-pressure liquid chromatography and found to consist of over 90%of Avarol. In addition, the biological activity of the compound in theblood was determined after extraction with ethyl acetate and estimatedin the L5178y cell system in vitro (Muller, W. E. G. et al.; Comp.Biochem. Physiol. 80C, 47-52; 1985). Using this system, the biologicalactivity of the compound, present in the blood, was determined andconverted on a gram basis. Using this approach the concentration in theblood was only 5-15% lower than that determined on the basis ofradioactivity.

Conclusion

(1) Avarol undergoes only a slight modification to abiologically-inactive form (not more than 15%) after circulation in thebody for 22 hours.

(2) Avarol has a long half-life (approximately 22 hours) after i.v.administration of anti-tumor concentrations.

(3) Avarol penetrates the blood-brain barrier. These properties ofAvarol in the body of rats appear to be highly favorable for an in vivoapplication of Avarol in man. On the basis of these data it is deducedthat, for the therapeutical application of Avarol in man, only 1-2injections are needed per day to warrant beneficial therapeutical doseswhich are in the range of 0.3 to 5 microgram/ml of blood.

Rationale: Interferon-Gamma Production Stimulation

Interferon-gamma is a mediator of T-lymphocyte immunity and is probablythe key-cell derived lymphokine that induces macrophages and otherpotential host defense cells to exert enhanced antimicrobial activityagainst both intracellular and extracellular pathogens. It isestablished that patients with AIDS and AIDS-related complex show animpaired gamma Interferon production (H. W. Murray et al.; The NewEngland Journal of Medicine 313; 1504-1510, 1985). The molecular reasonfor this manifestation is a profound impairment of antigen-inducedInterleukin-2 secretion (H. W. Murray; J. Clin. Invest 76: 1959-1964;1985). Under normal conditions in vivo, Interferon gamma is produced byT-lymphocytes, which have to be stimulated by Interleukin-2. Antigensfrom viruses such as vaccinia or herpes simplex can trigger Interferongamma secretion in leukocytes from immunized humans.

It is therefore advantageous that an anti-AIDS or anti-adult T-cellleukemia/lymphoma chemotherapeutic agent is provided with the ability toinduce gamma Interferon in T-lymphocytes. Avarol is such a compoundwhich combines both anti-AIDS and anti-adult T-cell leukemia/lymphomaactivity with the ability to induce Interferon gamma production. Theexperiments were performed in vitro, using peripheral human lymphocytes(buffy coat cells) in culture.

MATERIALS AND METHODS

Effector cells: Buffy coat cells were prepared by centrifugation onFicoll-Plaque gradient and washed two times. Subsequently the cells weredivided and adjusted to a concentration of 5×1,000,000 cells/ml.

Interferon measurement: Levels of Interferon were defined by anEnzymeimmunoassay (EIA). Interferon gamma: 96 well flat bottom plates(Greiner, Nuertingen) were coated with a monoclonal antibody recognizingInterferon gamma. 200 μl of the supernatant sample were added per welltogether with Interferon gamma-peroxidase coupled (50 μl), consisting ofthe same antibody as used for coating. After 24 hours of incubation theplates were washed with buffered salt solution containing 0.5% w/v Tween20™ detergent. The enzymatic reaction is completed by a preparationcontaining hydrogen peroxide substrate. The reaction is stopped within15 minutes by 100 μl of sulfuric acid per well. The amount of Interferonis defined by comparing the extinction of the test sample with astandard curve produced by known values of Interferon gamma.

Interferon: Interferon gamma was purchased from Hoffmann-La Roche,Grenzach, Germany.

Results

Buffy coat cells were incubated with different Avarol concentrations for0-96 hours. The results are summarized as follows:

    ______________________________________                                                 Titer of Interferon-gamma produced by                                Avarol   lymphocytes (Units/ml of supernatant)                                concentration                                                                          Incubation period (hours)                                            (μg/ml)                                                                             0        24      48      72    96                                    ______________________________________                                        0        2         5       3       7     4                                    0.2      4        17      24      26    21                                    0.5      4        23      74      89    118                                   1.0      3        20      52      62    73                                    ______________________________________                                         (The results are means of four parallel experiments; the standard             deviations are less than 10%)                                            

From these data, it is evident that Avarol induces significant andpronounced Interferon gamma in vitro at an optimal dose of 0.5 μg/ml.

Conclusion

Avarol has an additional beneficial therapeutic effect: Thehere-presented in vitro studies show that Avarol induces Interferongamma production in vitro and suggests that it acts also in vivo in thesame manner in adult T-cell leukemia/lymphoma patients.

In conclusion, from the foregoing, it is apparent that the presentinvention provides a novel method for the treatment and control of adultT-cell leukemia/lymphoma using Avarol and derivatives and/or precursorsand/or prodrugs thereof, and pharmaceutical compositions embodying theseactive ingredients for the said intended use, all having the foregoingenumerated characteristics and advantages.

It is to be understood that the invention is not to be limited to theexact details of operation, or to the exact compositions, methods,procedures or embodiments shown and described, as obvious modificationsand equivalents will be apparent to one skilled in the art, and theinvention is therefore to be limited only by the full scope which can belegally accorded to the appended claims.

I claim:
 1. A method of treating adult T-cell leukemia or lymphomacomprising the step of administering an effective anti-adult T-cellleukemia or lymphoma amount of a compound avarol to a living animal bodyin need of said treatment.